Single concentration confirmation of uHTS inhibitor hits of the thioesterase domain of fatty acid synthase via a fluorescence intensity assay

This study will focus on developing drug-like inhibitors/probes against fatty acid synthase, an enzyme that is essential for growth of solid tumors. Notably, FAS has only marginal importance in adults because dietary fat provides for normal physiology. The link between FAS and cancer was uncovered in 1994 when Frank Kuhajda found that the OA-519 antigen, a marker for poor prognosis in breast more ..

This study will focus on developing drug-like inhibitors/probes against fatty acid synthase, an enzyme that is essential for growth of solid tumors. Notably, FAS has only marginal importance in adults because dietary fat provides for normal physiology. The link between FAS and cancer was uncovered in 1994 when Frank Kuhajda found that the OA-519 antigen, a marker for poor prognosis in breast and prostate cancer, is actually fatty acid synthase. A number of subsequent immunohistochemical analyses showed that increased expression of FAS is a hallmark of all major cancers. The correlation between expression of FAS and poor prognosis strongly suggests that this enzyme is mechanistically linked to disease progression, providing a strong rationale for pursuing the development of FAS inhibitors.

The FAS protein contains six enzymatic domains and an acyl-carrier protein (ACP). The final enzymatic pocket is a thioesterase, which liberates the final product (palmitate) from its link to the ACP. It is the thioesterase domain of FAS which we plan to target here. To our knowledge, no thioesterase (TE) has ever been targeted for drug development. The goal of this high-throughput assay is to identify hit compounds for the FAS-TE domain. This is accomplished via an enzymatic reaction utilizing a fluorogenic substrate, O-methyl fluorescein heptanoate (OMFH).

The goal of this assay is to confirm hits in "uHTS identification of small molecule inhibitors of the thioesterase domain of fatty acid synthase via a fluorescence intensity assay", AID 602261.

Compounds that demonstrated a % activity >= 50% at 15 uM concentration in at least one replicate are defined as inhibitors of the reaction.To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Activity ScoringActivity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:a. If outcome of the primary screen is inactive, then the assigned score is 0b. If outcome of the primary screen is inconclusive, then the assigned score is 10c. If outcome of the primary screen is active, then the assigned score is 20Scoring for Single concentration confirmation screening is not applicable to this assay.d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay